A 1kb Plus ladder was used as a molecular size marker (M) with sizes indicated in base pairs. 75 Figure 3.10: Gel electrophoresis (2%) of rotavirus PCR product after concentration of . A 1 kb Plus ladder was used as a molecular size marker (M) with sizes indicated in base pairs. 77 Figure 3.12: Gel electrophoresis (2%) of rotavirus PCR product after concentration of.
INTRODUCTION
Examples of point source pollution include commercial and industrial companies, which often discharge wastes such as solvents and heavy metals from their operations (Gumbo et al Manders et al., 2009). As a result, the quality of the water sources with regard to virus contamination is not known (Bosch et al., 2008).
WATERBORNE PATHOGENS
The development of direct methods to monitor for human viral pathogens is therefore crucial as studies have also shown that rotaviruses are the most common cause of gastroenteritis worldwide and are responsible for the most severe form of viral gastroenteritis in humans (Gerba et al., 1996; Ahn et al., 2006; Mazari-Hiriart et al., 2009). The lower panel shows immunofluorescence images of Cryptosporidium ooc cysts and Giardia cysts (left) and a concentration-graded wet mount image stained with iodine of an Entamoeba cyst (Adapted from Bouzid et al., 2008).
WATERBORNE VIRUSES
RNA viruses
- Rotavirus
Rotavirus is one of the main causes of severe acute gastroenteritis worldwide and can infect infants, young children and adults, with infants and children having the most severe symptoms. The outermost layer of the capsid, which is required for cell attachment, membrane penetration, and cell entry, consists of two structural proteins (VP4 and VP7).
GENOME SEGMENTS
Of these 12 proteins (Figure 1.5), six are structural (VPs) and six are non-structural proteins (NSPs). The rotavirus capsid is also composed of three concentric protein layers that enclose the genome (Prasad and Estes, 2000).
ENCODED PROTEINS
The VP4 protein has been implicated in cell attachment, cell penetration, hemagglutination, neutralization and virulence, while VP7 may also play a similar role (Estes, 2001; Morris and Estes, 2001). Viral proteins three (VP3) and six (VP6) encode proteins required for RNA transcription and correct viral structure.
VIRION SCHEMATIC (Protein Locations)
RECONSTRUCTION
Norovirus
Norovirus is the prototype of the human calicivirus (Smit et al., 1997) and is classified under the genus noroviruses and the family Caliciviridae. Norovirus genomic nucleic acid alone could also be infectious (Haramoto et al., 2004; . ICTVdb, 2006).
Enterovirus
Enteroviruses are well described and known to cause meningitis, paralysis, rash, fever, myocarditis, respiratory disease and diarrhea in humans (van Regenmortel et al., 2000; Kubo et al., 2002). Enteroviruses are responsible for approximately 30 million cases of gastroenteritis per year in the United States, with children under the age of ten most affected (Ehlers et al., 2005).
Hepatitis A virus
41 with household bleach, and exposing it to heat higher than 85ºC for at least one minute also results in inactivation (Melnick, 1992; Nainan et al., 2006). The hepatitis A virus is one of the main causes of acute hepatitis and is distributed throughout the world (Villar et al., 2006). Hepatitis A virus infection is often asymptomatic among children, while symptoms are clearly visible in infected adults (Lemon, 1997; Hendrickx et al., 2008).
Overcrowding, poor sanitation and lack of a reliable clean water supply predispose people to hepatitis A infection (Nainan et al., 2006). Transmission of virus is highest 14 – 21 days after infection, corresponding to the period of peak shedding of virus and after the development of jaundice (Lemon, 1997; Gilroy et al., 2008). Tracing the contamination to the onions before they arrived in the United States illustrated the resiliency of the virus to survive and spread (Gilroy et al., 2008).
Vaccines are available that can reduce disease incidence and potentially eliminate transmission (WHO, 2000b; Pickering et al., 2006a; Nainan et al., 2006).
Adenovirus
- Mode of Transmission
However, the decrease in mortality can be attributed to the vaccination program launched by the Ministry of Health (DOH, 2005). Adenovirus infections can occur throughout the year, although adenovirus-associated respiratory disease is more prevalent in late winter (Pickering et al., 2006b). Adenovirus can be shed for long periods and can cause infections throughout the year, but respiratory diseases caused by adenovirus generally occur in winter, summer and early summer.
The spread of disease outbreaks associated with adenoviruses can also be prevented by good infection control practices such as regular hand washing (Horwitz, 1995; Foy, 1997; Pickering et al., 2006b). Adenovirus prevalence has been shown to be consistent worldwide with serotypes 40 and 41 accounting for between 38% and 100% of adenovirus serotypes (Enriquez, 1995; Moore et al., 2000). A respiratory infection caused by adenovirus can manifest as the common cold syndrome, pneumonia, croup or bronchitis.
In addition, adenovirus infections can cause serious complications in immunocompromised patients (Pickering et al., 2006b; Jones et al., 2007).
Aims of Study
Overview of Methodology
- Isolation and Concentration of Viruses
- Virus Detection Methods
- Water Samples for Pilot Study
- Concentration Techniques
- Concentration of Viruses by Silicon Dioxide (SiO 2 ) Method
- Concentration of viruses using Positively Charged Filters
- Concentration of viruses using Negatively Charged Filters
- Viral Extraction Techniques .1 TRIzol method
- QIAamp UltraSens Virus Kit (Qiagin)
- Reverse Transcription/cDNA Synthesis .1 Rotavirus cDNA synthesis
- Polymerase Chain Reaction (PCR) .1 Templates for PCR
- Amplification of adenovirus DNA
- Agarose Gel Electrophoresis
- Sequencing
- Sampling Sites
- Sample Collection
- Concentration Technique
- Concentration using Positively Charged Filters
- QIAamp UltraSens Virus Kit (Qiagen)
- Reverse Transcription/cDNA synthesis .1 Enterovirus cDNA synthesis
- Rotavirus cDNA synthesis
- Polymerase Chain Reaction (PCR) .1 Amplification of enterovirus cDNA
- Amplification of rotavirus cDNA
- Amplification of adenovirus DNA
- Agarose Gel Electrophoresis
Both methods minimized inhibitory substances when performing nucleic acid amplification by PCR (Kok et al., 2000). In addition, Fong et al. (2010) used the DNeasy tissue kit (Qiagen) to extract viral DNA from wastewater and reported that their goals were met. The reporter molecule can therefore be detected using fluorescence measurement procedures (van Pelt-Verkuil et al., 2008).
Other variations to the PCR protocol include booster PCR and time-release PCR (van Pelt-Verkuil et al., 2008). Site A - agricultural farming areas (Front site); Site B - nearest point to the informal settlement Kayamandi (On site) and Site C - substation in industrial area (After site) (Paulse et al., 2009). Based on results obtained in the pilot study (Section 2.2), the QIAamp Ultrasens Virus kit was used for the extraction of DNA and RNA from all samples collected along the Berg and Plankenburg rivers.
Sequences were identified using the Basic Local Alignment Tool (BLASTn) (Altschul et al., 1997) obtained from the National Center for Biotechnology Information (NCBI) website.
Introduction
- Viral extraction and concentration techniques
- Concentration of Viruses by Silicon Dioxide (SiO 2 ) Method
- Concentration of Viruses by Filtration
- Viral Nucleic Acid Extraction
- TRIzol Method for Nucleic acid Extraction
- QIAamp Ultrasens Virus Kit for nucleic acid extraction
- Polymerase Chain Reaction – Optimisation of Concentration and Extraction Methods
- PCR Results of Concentration and Extraction Methods
- Summary: Pilot Study
In contrast, the total extracted DNA in Figure 3.7 (B) showed improved resolution when the positively charged filter/QIAamp method (lane 3) and the mixed-ester filter/QIAamp method (lane 5) were used, while low-intensity smears were visible when the SiO2/QIAamp method (lane 2) and the negatively charged filter/QIAamp method (lane 4) were used. For the rotavirus samples extracted using the TRIzol method, Figure 3.8, rotavirus was not detected from cDNA when the oligo(dT)15 primer was used (lane 2), but a positive result of 346 bp was obtained when cDNA was prepared with a gene-specific primer (lane 3). For RNA samples extracted using the QIAamp Ultrasens Virus kit, the rotavirus PCR results illustrated in Figure 3.8 indicated that rotavirus was detected when cDNA was prepared using both the oligo(dT)15 (lane 4) primer and a gene-specific primer (lane 5) using the SiO2 concentration method.
Additionally, samples extracted using the QIAamp Ultrasens Virus kit (Figure 3.10) showed that rotavirus was detected when cDNA was prepared using oligo(dT)15 (lane 4) and with a gene-specific primer (lane 5). After concentration using positively charged filters, the polymerase chain reaction was also positive when DNA samples were extracted from adenovirus using the Roche High Pure PCR Model Preparation Kit (Figure 3.11, lane 2) and the QIAamp Ultrasens Virus Kit ( Figure 3.11, lane 3). In addition, for RNA samples extracted using the QIAamp Ultrasens Virus kit (Figure 3.12), no rotavirus was detected when cDNA was prepared using oligo(dT)15 primer (lane 4).
The PCR procedure also gave positive results when DNA samples were extracted from adenovirus using the Roche High Pure PCR Template Preparation kit (Figure 3.15, lane 2) and the QIAamp Ultrasens Virus kit (Figure 3.15, lane 3), after concentration of viral particles using non-charged mixed ester filters.
Environmental Water Samples Analysis
- Detection of Viral Antigens and Particles using the Positively Charged Filters The results for the Combi-Strip analyses, which tested for the presence of antigens, as well as
- Testing for Virus Presence using PCR .1 Rotavirus
- Enterovirus
Lane C is the rotavirus control while lanes 1–17 correspond to water samples collected over the 12 month sampling period from the Plankenburg River. Lane C is the rotavirus control while lanes 18–34 correspond to water samples collected over the 12 month sampling period from the Plankenburg River. Lane C is the rotavirus control while lanes 56–72 correspond to water samples collected over the 12-month-hourly sampling period from the Mountain River.
Lane C is an adenovirus control, while lanes 1-17 correspond to water samples collected from the Plankenburg River over a 12-month sampling period. Lane C is an adenovirus control, while lanes 18-34 correspond to water samples collected from the Plankenburg River over a 12-month sampling period. Nested PCR was used to detect enterovirus in river water samples collected from both river systems throughout the study period.
Samples collected from the Plankenburg River were labeled 1-36 (three samples per month for 12 months), while surface water samples collected from the Berg River were labeled 37-72 (three samples per month for 12 months).
LIMITATIONS
The absence of rotaviruses in our study was in contrast to data reported by other groups (Kittigul et al., 2005; Miagostovich et al., 2008) who found about 20% and 44% in river water, respectively. During this study, we could not determine any seasonal pattern for the distribution of the enteroviruses found. 2005) observed no seasonal pattern, possibly due to the mild temperatures of the autumn and winter months in South Africa.
RECOMMENDATIONS
Comparison of an immunochromatographic test for the simultaneous detection of rotavirus and adenovirus in stool. Three-step isolation method for sensitive detection of ent erovirus, putrefaction virus, hepatitis A virus and small round structured viruses in water samples. Detection of noroviruses in tap water in Japan by a new method for concentrating enteric viruses in large volumes of fresh water.
Three-step isolation method for sensitive detection of enterovirus, rotavirus, hepatitis A virus and small round structured viruses in water samples. Development of a virus concentration method and its application for the detection of noroviruses in drinking water in China. Determination of a universal nucleic acid extraction procedure for PCR detection of gastroenteritis viruses in fecal samples.
Detection of adenoviruses and rotaviruses in drinking water sources used in rural areas of Benin, West Africa.
